Human adenoviruses are non-enveloped icosahedral particles of −60 to 90 nM size. To date, 51 serotypes have been identified that are subdivided into six subgroups based on hemagglutination properties and sequence homology (Francki et al., 1991). The genome has a length of 34 to 36 kb and is flanked on both sites by inverted terminal repeat sequences (ITR). The virus infectious cycle is divided into an early and a late phase. In the early phase—(six to eight hours after infection), the virus is uncoated and the genome transported to the nucleus, after which the early gene regions E1-E4 become transcriptionally active.
The early region-1 (E1) contains two transcription regions named E1A and E1B. The E1A region encodes two major proteins that are involved in modification of the host-cell cycle and activation of the other viral transcription regions (reviewed by Russell, 2000). The E1B region encodes two major proteins, 19K and 55K, that prevent, via different routes, the induction of apoptosis resulting from the activity of the E1A proteins (Rao et al., 1992; Yew and Berk, 1992; reviewed in Shenk, 1996). In addition, the E1B-55K protein is required in the late phase for selective viral mRNA transport and inhibition of host protein expression (Pilder et al., 1986). Early region-2 (E2) is also divided in an E2A and E2B region that together encode three proteins, DNA binding protein, viral polymerase and pre-terminal protein, all involved in replication of the viral genome (reviewed by van der Vliet, 1995). The E3 region is not necessary for replication in vitro but encodes several proteins that subvert the host defense mechanism towards viral infection (reviewed by Horwitz, 2001). The E4 region encodes at least six proteins involved in several distinct functions related to viral mRNA splicing and transport, host-cell mRNA transport, viral and cellular transcription and transformation (reviewed by Leppard, 1997).
The late proteins necessary for formation of the viral capsids and packaging of viral genomes, are all generated from the major late transcription unit (MLTU) that becomes fully active after the onset of replication. A complex process of differential splicing and polyadenylation gives rise to more than 15 mRNA species that share a tripartite leader sequence. The early proteins E1B-55K and E4-Orf3 and Orf6 play a pivotal role in the regulation of late viral mRNA processing and transport from the nucleus (reviewed in Leppard, 1998).
Packaging of newly formed viral genomes in pre-formed capsids is mediated by at least two adenoviral proteins, the late protein 52/55K and an intermediate protein IVa2, through interaction with the packaging sequence located at the left end of the genome (Grable and Hearing, 1990; Gustin and Imperiale, 1998; Zang et al., 2001). A second intermediate protein, pIX, is part of the capsid and is known to stabilize the hexon-hexon interactions (Furcinitti et al., 1989). In addition, pIX has been described to transactivate TATA-containing promoters like the E1A promoter and MLP (Lutz et al., 1997).
Due to the extensive knowledge of the viral biology and the high efficiency of nuclear delivery after entry into cells, adenoviruses have become popular tools for gene delivery into human cells. In addition, adenoviral vectors are stable and can be produced relatively easy at a large scale. In most cases, vectors are deleted for at least the E1 region, which renders them replication deficient. Production of E1-deleted vectors based on subgroup C serotypes Ad5 or Ad2 is achieved in E1-complementing cell lines such as 293 (Graham et al., 1970), 911 (Fallaux et al., 1996) and PER.C6™ (Fallaux et al., 1998). As disclosed in U.S. Pat. No. 5,994,128, vectors and cell lines need to be carefully matched to avoid generation of replication-competent adenoviruses through homologous recombination between adenovirus sequences in the cell line and the vector. Thus, PER.C6™ cells and matched adenoviral vectors provide a preferred system for the production of group C adenoviral vectors (Fallaux et al., 1998). The deletion of E1 sequences provides space for the introduction of foreign genes in the viral vector. Since the maximum size of Ad5 genomes that can be incorporated into virions is limited to about 105% of the wild-type length, E1-deleted viruses can accommodate approximately 4.8 kb of foreign DNA (Bett et al., 1993).
The maximum packaging capacity in virions that lack pIX is reduced to approximately 95% of the normal genome length (Ghosh-Choudhury et al., 1987). This is most likely caused by the reduced stability of pIX— (“pIX-minus”) virions. The deficiency in pIX-minus mutant Ad5 can be complemented by episomal expression of pIX in a packaging cell line used for producing viruses (Caravokyri et al., 1995).
Although the serotypes Ad5 and Ad2 are most commonly used as gene transfer vectors, other serotypes may have preferred characteristics that make them more useful as a therapeutic or prophylactic tool. Subgroup B viruses Ad35 and Ad11, for example, are much less prone to neutralization by human sera than Ad5 and Ad2 viruses (disclosed in WO 00/70071). Neutralization of adenoviral transfer vectors diminishes transduction efficiency in vivo. Furthermore, the infection efficiency of antigen presenting cells, like dendritic cells, by recombinant viruses carrying the fiber of Ad35 was found to be greatly enhanced in vitro compared to Ad5 viruses (WO 00/70071, WO 02/24730). Thus, Ad35-based vectors combine highly improved infection efficiency with low neutralization in human sera, making such vectors suitable for vaccination purposes.
Generation and propagation of fully E1-deleted Ad35-based vectors is possible using the technology discussed below. However, careful analysis of a variety of recombinant Ad35-based vectors has revealed that such vectors are less stable, i.e., can contain less foreign DNA compared to the Ad5-based vectors. In the current patent application, means and methods are presented to overcome this problem.
In addition, there is a need to further develop the presently available technology for adenoviruses that have broader serotype utility. Existing packaging cell lines typically comprise E1-encoded proteins derived from adenovirus serotype 5. Examples of such “standard” packaging cell lines are 293, 911 and PER.C6™. Attempts to produce vectors derived from other serotypes on these standard packaging cell lines have proven arduous, if not unsuccessful. Occasionally, some production is seen, depending on the particular serotype used. However, the yields of recombinant adenovirus vectors derived from adenovirus subgroups other than subgroup C, produced on cell lines transformed and immortalized by E1 from Ad5, is poor. In a paper by Abrahamsen et al. (1997), improved plaque purification of an E1A-deleted adenovirus serotype 7 vector (subgroup B) was observed on 293 cells comprising E4-orf6 derived from adenovirus serotype 5, as compared to 293 cells lacking the E4-orf6 sequence from Ad5. However, a problem was encountered with the stability of the vector as unexpected recombinations were observed in plaque-purified stocks. An additional problem was encountered with wild-type adenovirus contamination during production. Moreover, for large-scale production of adenoviruses, it is not useful to co-transfect E4-orf6 to obtain titers that are high enough for application. One option for growing such adenoviruses is to provide cells with the E4-orf6 gene stably integrated into the genome of the complementing/packaging cell line. Such cells have been described in the art (e.g. WO 96/22378). A disadvantage of that system is the fact that new stable cell lines have to be generated and numerous selection rounds have to be performed before stable and proper cells have been generated. This process is laborious and time-consuming. In general, it can be stated that generation and propagation of adenoviruses from serotypes other than serotype 5 (subgroup C), such as subgroup B viruses, have proven to be difficult on Ad5-complementing cells. As has been disclosed by the applicants in WO 00/70071, recombinant viruses based on subgroup B virus Ad35 can be made by co-transfection of an expression construct containing the Ad35-early region-1 sequences (Ad35-E1). Furthermore, Ad35-based viruses that are deleted only for E1A sequences and not for E1B were shown to replicate efficiently on PER.C6™ cells, suggesting that the E1A proteins of Ad5 are able to complement the Ad35-E1A functions (applicant's application WO 02/40665). Moreover, the experiments show that lack of Ad35-E1B results in poor yields on Ad5-complementing cells. WO 00/70071 also discloses cell lines for the production of E1-deleted non-group C adenoviral vectors by further modifying cell lines that are capable of complementing adenovirus serotype 5. WO 00/70071 further suggests that one should establish new cell lines harboring Ad35-E1 sequences for the complementation of recombinant adenovirus serotype 35 vectors lacking the E1 region (see also WO 02/40665). However, as also discussed above, if one desires to apply a specific serotype for a specific need, one would have to establish a new cell line for every specific serotype or one would have to modify the available cell lines that can complement adenovirus serotype 5 for complementation of the serotype of interest. It would clearly be advantageous to use the established cell lines that are available in the art and not to modify these and use them for the production of all other, non-Ad5 serotypes, applying the established and efficient methods known in the art.
It is concluded that there still exists a need for a production system to produce useful yields of adenovirus serotypes that are different from the serotypes of subgroup C.
Furthermore, there is still a need for suitable packaging systems comprising convenient packaging cells and recombinant subgroup B adenoviruses that are stable and can be propagated on such packaging cells.